BiOWiSHTM – Aqua is a blend of preserved multi-bacteria culture with the capability of denitrification. If an anaerobic nitrate rich activation procedure is used instead of the standard aerobic activation procedure, the denitrification rate is increased by 28 percent under the conditions of 30°C, 1C:1N, 200mg/L of carbon, and 200mg/L nitrogen.

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The highly specific recognition processes between biomolecules mediate various crucial biological processes. Uncovering the molecular basis of these interactions is of great fundamental and applied importance. This research work focuses on understanding the interactions of several biomolecular recognition systems and processes that can provide fundamental information to aid in the rational design of sensing and molecular recognition tools. Initially, a reliable and versatile platform was developed to investigate biomolecular interactions at a molecular level. This involved several techniques, including biomolecule functionalization to enable attachment to self-assembled monolayers as well as atomic force microscopy (AFM) based force spectroscopy to uncover the binding or rupture forces between the receptor and ligand pairs. It was shown that this platform allowed determination of molecular binding between single molecules with a high specificity. The platform was further adapted to a general sensing formulation utilizing a group of flexible and adaptive nucleic acid recognition elements (RNA and DNA aptamers) to detect specific target proteins. Investigation of interactions at the molecular level allowed characterization of the dynamics, specificity and the conformational properties of these functional nucleic acids in a manner inaccessible via traditional interaction studies. These interactions were then adapted to aptamer-based detecting methods that at the ensemble or bulk scale, specifically taking advantage of mechanisms uncovered in the biophysical study of this system. A quartz crystal microbalance (QCM) was used to detect protein targets at the bulk level and the affinities and binding kinetics of these systems were analyzed. Along with AFM-based force spectroscopy, ensemble-averaging properties and molecular properties of these interactions could be correlated to contribute to bridging the gap across length scales. Finally, more broadly applicable sensing platform was developed to take advantage of the unique properties of aptamers. DNA was employed both as a carrier and as a molecular recognition agent. DNA was used as a template for nanoconstruction and fabricating unique shapes that could enhance the aptamer-based molecular recognition strategies. With aptamers tagged to distinct nanoconstructed DNA, a novel shape-based detecting method was enabled at the molecular level. The results demonstrated that this is a flexible strategy, which can be further developed as ultrasensitive single molecule sensing strategy in complex environments.

The recently proposed bioassay procedure that is based on the substrate induced quenching (SIQ) of an indicator fluorescence for the measurement of analyte concentrations is evaluated. In this type of assay a enzynatic reaction and a fluorescence quenching interaction are coupled together. Typically, an appropriate dehydrogenase enzyme reduces or oxidises the nicotinamide adenine dinucleotide cofactor. The change in the concentration of NADH results in variations in the excited fluorophore population as observed through fluorescence intensity. This latter aspect is used to monitor substrate (analyte) concentrations. Results on the investigation of the substrate induced quenching bioassay method and possibilities of using it as the basis of (i) a novel enzyme bioassay technique and (ii) a novel bioprobe format are presented. Ethanol was chosen as the model analyte, and a new assay procedure for its measurement was developed. A generic theoretical relation is discussed for the observed assay kinetics of substrate induced quenching (SIQ) and a model is described that includes the effects due to dynamic/static quenching of the fluorophore by either the enzyme substrate or product. The validity of the derived model is shown by comparison with experimental results for a SIQ based ethanol assay. The option of running the dehydrogenase reaction so as to consume NADH rather than generate it is also investigated. In order to demonstrate this approach acetaldehyde was chosen as the model analyte, and a assay procedure for its measurement was developed. The potential of the SIQ technique for incorporation into biosensor based upon a 'reservoir' format was demonstrated through the development of custom optical instrumentation and resevoir flowcell. Applicability of the SIQ technique to other biosensor formats such as flow-injection analysis and 'dry reagent' technology is discussed. The overall applicability of the SIQ technique is assessed through the generation of a number of SIQ assays on the following substrates: ethanol, glucose, glucose-6- phosphate, L-glutamic acid, isocitric acid, acetaldehyde, pyruvic acid, ot-ketoglutaric acid, and oxalacetic acid.

Neuerliche Fortschritte im Verständnis biomolekularer Motoren rücken ihre Anwendung als Nanomaschinen in den Bereich des Möglichen. So könnten sie zum Beispiel als Nanoroboter arbeiten, um in molekularen Fabriken kleine – aber dennoch komplizierte – Strukturen auf winzigen Förderbändern herzustellen, um Netzwerke molekularer Nanodrähte und Transistoren für elektronische Anwendungen zu assemblieren oder sie könnten in adaptiven Materialien patrouillieren und diese, wenn nötig, reparieren. In diesem Sinne besitzen biomolekulare Motoren das Potenzial, die Basis für die Konstruktion, Strukturierung und Wartung nanoskaliger Materialien zu bilden. / Recent advances in understanding how biomolecular motors work have raised the possibility that they might find applications as nanomachines. For example, they could be used as molecule- sized robots that work in molecular factories where small, but intricate structures are made on tiny assembly lines, that construct networks of molecular conductors and transistors for use as electrical circuits, or that continually patrol inside “adaptive” materials and repair them when necessary. Thus biomolecular motors could form the basis of bottom-up approaches for constructing, active structuring and maintenance at the nanometer scale.

Lanksti vanadžio pentoksido kserogelio struktūra, susidedanti iš vanadžio-deguonies sluoksnių, atskirtų vandens molekulėmis, leidžia įterpti tarp sluoksnių ne tik atskirų elementų jonus, bet ir molekulinius darinius. Šio darbo tikslas yra sukurti naujų vanadžio – gliukozės pagrindu metalo-bioorganinių junginių sintezės technologiją, ištirti jų paviršiaus dangas, Rentgeno fotoelektronų spektrus bei cheminę sudėtį. Darbe trumpai aprašyti Skenuojančio elektronus, atominių jėgų ir Rentgeno fotoelektronų spektroskopijos metodų pagrindai, vanadžio oksidų savybės, V2O5 gelių struktūra ir sintezės metodai. V2O5/xGL junginiai buvo gamina trimis etapais: vanadžio pentoksido kserogelio sintezė, naudojant zolis-gelis technologija; gliukozės (C6H12O6) įterpimas į kserogelio struktūrą ir V2O5/xGL kserogelio atkaitinimas prie 580 0C, siekiant pašalinti chemiškai surišta vandenį. Gauta V2O5/xGL junginiai buvo tiriami Rentgeno fotoelektronų spektroskopijos, skenuojanti elektronus mikroskopijos, atominių jėgų mikroskopijos metodais. Vanadžio-deguonies ir anglies sričių RFS spektrų pokyčiai, įvedus į gryno vanadžio pentoksido kserogelio struktūrą gliukozės molekules, leidžia tvirtinti, kad mes gavome ne paprastą vanadžio ir gliukozės mišinį, o buvo susintetintas V2O5/GL junginys, kuriame susidarė tam tikri cheminiai ryšiai tarp V2O5 ir C6H12O6 molekulių. Tuom mes įrodome, kad gliukozės molekulę (C6H12O6) įterpėme į vanadžio pentoksido struktūrą. Gauti rezultatai leidžia tvirtinti, kad... [toliau žr. visą tekstą] / Flexible structure of vanadium pentoxide xerogel, consisting of vanadium-oxygen layers, separated by water molecules, allows to insert ions of separate elements, as well as molecular formations, between the layers. The purpose of this work is to produce a new vanadium – glucose based metal – bioorganic compounds fusion technology, to explore the surface of coating, X – ray photoelectron spectra and chemical composition. In this work the basics of the Scanning electron, Atomic force and X-ray photoelectronic spectroscopy methods, features of vanadium oxides, structure of V2O5 and synthesis methods are briefly described. V2O5/xGL compounds was produced in three stages: synthesis of vanadium pentoxide xerogel, using zol-gel technology; inserting glucose into the structure of xerogel and annealing of the obtained vanadium hydrochinon xerogel at 580°C, in order to remove the chemically linked water. The obtained V2O5/xGL was analyzed using the Scaning electron, Atomic force and X-ray photoelectronic spectroscopy methods. Vanadium-oxygen and carbon XPS spectra of the fields changes shows that using of sol-gel technology methods, we synthesized V2O5/xGL compounds.

The field of nanotechnology has enabled scientists to perform fascinating engineering manipulations of biological substrates. Systems of DNA are now able to perform algorithmic computations by way of constructing biological modules composed of DNA macromolecules and using laboratory techniques available to biological sciences. The tile assembly model is an established model of biomolecular computing: using properties of DNA macromolecules to define constructions of self-assembling biological systems. The existing tile assembly model uses the concept of DNA tiles conceptually shaped as squares and exposes the tiles to carefully controlled biological conditions. The result is that under this process we can design and create these systems to compute solutions to algorithmic problems. Hexagons are the only two-dimensional regular polygon other than squares that can tile a plane infinitely leaving no space uncovered, where only translations of the initial polygon is allowed. Therefore hexagon-shaped DNA tiles can be defined to cover a planar surface, with the notable difference of six adjacent tiles per position versus the four adjacent neighbours in traditional four sided tiles. In this thesis, we will define a generalization of the tile assembly model that supports six-sided DNA tiles, in addition to the traditional four sides. We will introduce a problem known as the 0-1 Knapsack problem that is currently unsolved with square tiles. Moreover, a solution to the problem was attempted by tile assembly model researchers, however we show there is an error in their solution. After we analyze their solution and discover the shortcomings of square tiles under those constraints, we then show this fault is not applicable to hexagon tiles. Therefore, we show that the 0-1 Knapsack problem is solvable using hexagon shaped tiles.

<p>Capillary electrophoresis is a high resolution microseparation technique that is increasingly being recognized as a physical tool to characterize biomolecular interactions, where dynamic complexation of analytes with discrete additives is used to resolve complex mixtures of solutes, including enantiomers. Despite the wide interest in developing high-throughput screening platforms for drug discovery or disease prognosis, little emphasis has been placed on enhancing "pre-analysis steps" that are often the most crucial component determining the overall performance of a method. Off-line sample pretreatment protocols for complex biological samples are often time-consuming and not amenable for automation. The major goal of this thesis is the development of a single-step analytical platform by CE for targeted metabolites that integrate several different sample pretreatment processes during separation, which can also be used to characterize the thermodynamic parameters associated with covalent and non-covalent interactions. Two distinct projects in this thesis have been examined involving boronic acid-polyol and protein-cyclic nucleotide interactions that illustrate the concept of integrating sample pretreatment with chemical analysis based on dynamic complexation-capillary electrophoresis.</p> <p>The first project consists of a new strategy for enhancing target selectivity when using 3-nitrophenylboronic acid as an electrokinetic probe in dynamic complexation-capillary electrophoresis. The differential migration of ternary boronate ester complexes permits the selective analysis of micromolar levels of UV-transparent polyol stereoisomers in urine samples that is applicable to single-step screening of in-born errors of sugar metabolism, such as galactosemia. In the second project, the impact of ligand binding on protein stability is assessed by dynamic ligand exchangeaffinity capillary electrophoresis with laser-induced native fluorescence detection. This is a convenient yet rapid format for comparative thermodynamic studies of a regulatory subunit of protein kinase involving different cyclic nucleotide analogues without off-line sample pretreatment, since ligand exchange and protein unfolding processes are integrated incapillary during electromigration.</p> / Thesis / Master of Science (MSc)

Molecular dynamics (MD) simulations are routinely used to study the structure and function of biological molecules. However the accuracy and duration of these simulations are constrained by their computational costs, thus limiting the ability to accurately simulate systems of realistic sizes over biologically relevant time periods. The two most computationally demanding steps in these simulations are (1) determining the charge state of ionizable sites in biomolecules, which is a key input to the simulation, and (2) calculating long range electrostatic interactions during the simulation. Presented here are two novel methods, the <i>direct interaction approximation (DIA)</i> and the <i>hierarchical charge partitioning (HCP) approximation</i>, for speeding up each of these two computations. The average charge state of ionizable sites in biomolecules can be calculated as the statistical average over all possible (2^N) microstates for a molecule, where N is the number of ionizable sites. In general this computation scales exponentially as O(N² 2^N). The DIA is an O(²) approximation for calculating the average charge state of ionizable sites. For each site, the DIA treats direct interactions (interactions involving the site of interest) <i>exactly</i>, while using an <i>average</i> value for indirect interactions (interactions not involving the site of interest). The DIA was tested on two problems. The computation of thermal average properties for the 2-D Ising model of ferromagnetism, and the average charge state of ionizable residues in biomolecules. Compared to the commonly used non-deterministic Monte Carlo method, for the same computational cost, the deterministic DIA was found to be at least as accurate, as measured by RMS error relative to the exact computation. Thus, the DIA may be a practical alternative to the Monte Carlo method for some problems. In atomistic MD simulations, the computation of long range electrostatic interactions, scale as O(<i>n</i>²), where <i>n</i> is the number of atoms. For most biologically relevant timescales the simulations involve 10^12–16 simulation steps. Thus, the computational cost of long range interactions become the limiting factor in the size and duration of MD simulations. The HCP is an O(<i>n</i> log <i>n</i>) approximation for computing long range electrostatic interactions. The approximation is based on multiple levels of natural partitioning of biomolecular structures into a hierarchical set of components. For components that are far from the point of interest, the charge distribution for each component is approximated by a much smaller number of charges. For nearby components, the HCP uses the full set of atomic charges. For large structures the HCP can be several orders of magnitude faster than the exact pairwise O(<i>n</i>²) all-atom computation. For a representative set of structures, the accuracy of the HCP is comparable to the industry standard explicit solvent particle mesh Ewald (PME), and is in general more accurate than the spherical cutoff method. And, unlike the PME, the DIA can be easily extended to implicit solvent GB models. 50 ns implicit solvent simulations for a representative set of four biomolecules suggests that the HCP could be a practical alternative for implicit solvent simulations, and preferable to the cutoff based method. The HCP is available for general use in the open source MD software, NAB within AmberTools. / Ph. D.

During the past decade, many successful deterministic models of macromolecular regulatory networks have been built. Deterministic simulations of these models can show only average dynamics of the systems. However, stochastic simulations of macromolecular regulatory models can account for behaviors that are introduced by the noisy nature of the systems but not revealed by deterministic simulations. Thus, converting an existing model of value from the most common deterministic formulation to one suitable for stochastic simulation enables further investigation of the regulatory network. Although many different stochastic models can be developed and evolved from deterministic models, a direct conversion is the first step in practice. This conversion process is tedious and error-prone, especially for complex models. Thus, we seek to automate as much of the conversion process as possible. However, deterministic models often omit key information necessary for a stochastic formulation. Specifically, values in the model have to be scaled before a complete conversion, and the scaling factors are typically not given in the deterministic model. Several functionalities helping model scaling and converting are introduced and implemented in the JigCell modeling environment. Our tool makes it easier for the modeler to include complete details as well as to convert the model. Stochastic simulations are known for being computationally intensive, and thus require high performance computing facilities to be practical. With parallel computation on Virginia Tech's System X supercomputer, we are able to obtain the first stochastic simulation results for realistic cell cycle models. Stochastic simulation results for several mutants, which are thought to be biologically significant, are presented. Successful deployment of the enhanced modeling environment demonstrates the power of our techniques. / Master of Science